The innate immune system that defends mammals from many Gram-negative bacteria is triggered when host cells sense the lipid A moiety of the bacterial cell wall lipopolysaccharide (LPS). The resulting inflammatory response then usually kills the bacteria. LPS-induced inflammation can also be harmful, however, and animals have numerous ways to limit its magnitude and duration. One important control mechanism is a highly conserved lipase, acyloxyacyl hydrolase (AOAH), that selectively removes from LPS the fatty acyl chains that are required for sensing by MD-2-TLR4, the mammalian LPS receptor. Mice that do not make AOAH (Aoah''') respond to low doses of LPS by producing large quantities of antibodies, developing striking liver enlargement, and maintaining prolonged endotoxin tolerance (a form of immunosuppression). LPS that is not deacylated by AOAH remains stimulatory for weeks in vivo. We now request continuing support to test the hypothesis that AOAH prevents prolonged reactions to LPS and Gramnegative bacteria in both local and systemic in vivo compartments. In Specific Aim 1, we will find out how LPS deacylation by phagocytes in a subcutaneous tissue bed regulates its ability to interact with a second LPS-responsive target cell, the B lymphocyte. These studies should provide the first quantitative information about how LPS processing by phagocytes prevents activation of other cells in extravascular tissue spaces. In Specific Aim 2 we shall find out where LPS deacylation occurs in the liver, the organ that normally detoxifies most bloodborne endotoxin, and define the cellular basis for the persistent sinusoidal endothelial activation that LPS induces in Aoah'A mice. These experiments should indicate how AOAH prevents prolonged LPS-induced inflammation in the intravascular (systemic) compartment. In Specific Aim 3, we shall use complementary strategies to find out whether increasing LPS deacylation in vivo, either by providing exogenous AOAH or by enhancing its endogenous expression, is likely to benefit animals with serious Gram-negative bacterial infections. In addition to defining the enzyme's role in controlling responses to LPS in vivo, the proposed studies should break new ground in understanding the "recovery" phase of Gram-negative bacterial infections, an important yet understudied aspect of bacterial pathogenesis and host defense.